Cis packaging element of single-chain monopole DNA recombinant adeno-associated virus, spAAV vector and application

Abstract

The invention provides a plurality of cis packaging elements, ITR (internal transcriptase), DtrsA 'B', trsA 'B', trsA ', DttRR and ttRR which are applied to packaging of single-chain monopole DNA (deoxyribonucleic acid) recombinant adeno-associated virus vectors (spAAV, spAAV), and the single-chain monopole DNA recombinant adeno-associated virus vectors produced by packaging. The cis packaging elements, the ITR, the DtrsA' B ', the trsA' B ', the trsA', the DttRR and the ttRR are applied to packaging of the single-chain monopole DNA recombinant adeno-associated virus vectors. The cis packaging element comprises a Rep binding site (RBS) and a Rep cutting site (tr), and the cis packaging element comprises a Rep binding site (RBS) and a Rep cutting site (tr), and the Rep binding site (RBS) and the Rep cutting site (tr) are arranged on the cis packaging element. The cis packaging element can package spAAV genomes into AAV capsid protein, the cis packaging element is arranged at the 5'end for packaging the single-stranded monopole DNA, and Rep protein packages the single-stranded monopole DNA into the AAV capsid protein through recognition and cutting. The titer and cell transfection performance of spAAV virus particles produced by packaging of the cis packaging elements are equivalent to those of traditional rAAV virus particles.

Description

Technical Field

[0001] The present invention relates to the field of gene technology, in particular to a cis packaging element of a single-stranded monopolar DNA recombinant adeno-associated virus, a spAAV vector and applications. Background Art

[0002] Gene therapy is aimed at genetic mutations caused by gene mutations, abnormal gene expression, etc., and can be treated, prevented or alleviated by providing patients with corrective genetic material, including prevention of genetic diseases caused by gene defects, abnormal regulation or abnormal expression, such as diseases caused by insufficient or overexpression of genes, malignant tumors, etc. At present, gene delivery vectors mainly include viral gene vectors and non-viral gene vectors. Among the many available viral-derived gene vectors, for example, recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc., recombinant adeno-associated virus (rAAV) gene vectors are becoming more and more popular.

[0003] Adeno-associated virus (AAV) belongs to the Parvoviridae family. It has become the most promising gene vector for gene therapy due to its advantages such as non-pathogenicity, low immunogenicity, and broad-spectrum infectivity. AAV is a non-enveloped single-stranded DNA virus that carries a linear single-stranded DNA genome of about 4.7kb. There is a 145bp inverted terminal repeat (ITR) at each end of the AAV DNA genome, in which the 125 bases near the end form a long palindromic structure, which can be folded by base complementary pairing, so that the ITR presents a T-shaped hairpin structure. The positive and negative strand DNA genomes carrying wild-type inverted terminal repeat sequences are packaged into the AAV virus capsid with the same probability, thereby producing the same number of positive or negative strand DNA AAV virus particles. AAV virus particles infect cells, enter the cell nucleus, uncapsidate, and release the AAV genome into the cell nucleus. The ITRs at both ends of the AAV genome can fold to form a T-shaped hairpin structure, and its 3′ end will serve as a primer for DNA synthesis to synthesize the second strand to form a double-stranded DNA molecule, thereby initiating the expression of the genes carried in the AAV genome. The positive and negative strands of the AAV genome can also form double-stranded DNA molecules to express genes through complementary pairing.

[0004] At present, it is generally believed in the art that the ITRs at both ends of the AAV DNA genome contain basic information for AAV DNA replication, packaging, integration, and rescue. Based on this, the rAAV packaging systems used all use the ITR of AAV as the basic packaging element, placed at both ends of the exogenous gene DNA that needs to be packaged into the rAAV capsid, and package the exogenous gene DNA into the rAAV vector. Such rAAV vectors have defects in the application of gene delivery, gene manipulation, and gene editing. For example, single-polarity single-stranded DNA cannot be delivered to perform single-stranded DNA gene editing; it is difficult to design rAAV vectors that produce short-term gene expression for short-term gene expression, such as delivering nuclease genes for gene editing, which causes off-target risks and cytotoxicity due to long-term gene expression; it is difficult to achieve single-stranded gene pairing to express large genes; it is impossible to deliver long oligodeoxynucleotides to carry out small nucleic acid gene therapy gene regulation, etc. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a cis packaging element of a single-stranded monopolar DNA recombinant adeno-associated virus, a spAAV vector and an application thereof, wherein the spAAV genome before packaging has a cis packaging element for packaging AAV DNA at the 5′ end of its linear single-stranded DNA. The vector for packaging the single-stranded monopolar DNA recombinant adeno-associated virus delivers a single-polarity single-stranded DNA, thereby performing single-stranded DNA gene editing, and further achieving single-stranded gene pairing to express large genes and deliver long oligodeoxynucleotides, so as to carry out small nucleic acid gene therapy gene regulation, etc.

[0006] To achieve the above objectives, the present invention provides a variety of cis packaging elements (DNA cis elements) of single-stranded monopolar DNA recombinant adeno-associated viruses; the cis packaging elements contain a Rep binding site (RBS) and a Rep cleavage site (trs); the cis packaging elements are capable of packaging the spAAV genome into the AAV capsid protein, including when the cis packaging element is placed at the 5′ end of the single-stranded monopolar DNA, the Rep protein packages the single-stranded monopolar DNA into the AAV capsid protein by recognizing and cleaving.

[0007] The cis packaging element of the spAAV vector includes at least one of ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR. When the cis packaging element is placed at the 5′ end of the packaging DNA, the Rep protein will recognize and cut, and the single-stranded monopolar DNA chain will be packaged into the AAV virus capsid. The spAAV genome before packaging has a linear open end (except for the use of the ITR cis packaging element), and its 5′ end only contains the cis packaging element, and the 3′ end does not contain any packaging-related sequence.

[0008] That is, the spAAV vector formed by packaging with the above-mentioned cis packaging element contains only DNA molecules of a single polarity, that is, a spAAV vector of a positive-strand DNA molecule, or a spAAV vector of a negative-strand DNA molecule, and is not a mixture of the two. Only when the cis packaging element is placed at the 5′ end of the upper and lower strands of the positive and negative complementary DNA at the same time, because there are cis elements upstream and downstream of the plasmid, the packaged AAV has both positive single-stranded genomes and negative single-stranded genomes, and can simultaneously package and produce rAAV virus particles with positive-strand DNA molecules and rAAV virus particles with negative-strand DNA molecules. Mixed products. The spAAV vector can advantageously deliver single-stranded DNA with linear open ends (except for the use of ITR cis packaging elements) of the same polarity (i.e., unipolar), achieving unipolar single-stranded DNA delivery (positive or negative strands).

[0009] Specifically, the cis packaging elements selected in the present invention all contain a Rep binding site and a Rep cleavage site (terminal resolution site, trs).

[0010] To achieve another purpose, the present invention also provides a spAAV vector, which is obtained by packaging the spAAV genome by providing the cis packaging element provided by the above technical solution; including AAV capsid protein and the spAAV genome packaged by the AAV capsid protein; wherein the spAAV genome packaged by the AAV capsid protein originates from the spAAV genome before packaging; the spAAV genome has a spAAV vector of positive-strand DNA, or a spAAV vector of negative-strand DNA, and the two are not produced at the same time. Only when the cis packaging element is placed at the 5′ end of the upper and lower chains of the positive and negative complementary DNA at the same time, a mixed product of rAAV virus particles with both positive-strand DNA molecules and rAAV virus particles with negative-strand DNA molecules can be packaged and produced simultaneously.

[0011] Furthermore, the method for obtaining the spAAV vector includes transforming the spAAV transgenic plasmid, the Cap expression plasmid, and the Rep expression plasmid into Escherichia coli competent cells respectively; after at least one round of blue-white screening, picking out white colonies, amplifying and extracting the recombinant bacmid; transfecting insect cells with the recombinant bacmid to produce a recombinant baculovirus; and, extracting the recombinant baculovirus, and infecting insect cells with the recombinant baculovirus, thereby obtaining the spAAV vector.

[0012] Based on the spAAV vector provided above, a method for delivering exogenous nucleic acid to a cell is realized, that is, the spAAV vector is contacted with a cell and infects the cell, and the genome of the spAAV vector comprises a gene expression frame of the exogenous nucleic acid and / or other optional DNA sequences; the spAAV vector is integrated into the cell genome, homologous or non-homologous recombination occurs, and the exogenous gene expression is generated;

[0013] Preferably, the genome of the spAAV vector comprises the positive-strand single-stranded DNA sequence and / or the negative-strand single-stranded DNA sequence of the exogenous gene.

[0014] Preferably, the exogenous nucleic acid comprises a nucleic acid sequence of an exogenous gene or a nucleic acid sequence of a non-exogenous gene.

[0015] The spAAV vector provided by the above technical solution can be applied to gene expression and / or gene therapy and / or gene editing and / or gene regulation or related products.

[0016] The technical effects of the technical solution of the present invention are as follows:

[0017] 1. The spAAV genome before packaging provided by the technical solution of the present invention has a cis packaging element at the 5′ end of its linear single-stranded DNA. In particular, the spAAV vector formed by the spAAV genome before packaging in the present invention has only one polarity, that is, either a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time. In addition, the titer and cell transfection performance of the spAAV virus particles produced by packaging are comparable to those of traditional rAAV virus particles.

[0018] 2. The spAAV virus particles formed by the packaging according to the present invention can advantageously deliver monopolar, single-stranded DNA of the same polarity with linear open ends (except for the use of ITR cis packaging elements). Since it is only monopolar, single-stranded DNA of the same polarity and there is no 3′ITR structure that can be used as a DNA replication primer, spAAV DNA can only exist in the form of single-stranded DNA in cells, so it can be used for gene integration, gene editing, promoting efficient DNA pairing to express large genes and deliver long oligodeoxynucleotides, so that it can carry out small nucleic acid gene therapy and the like.

[0019] 3. The present invention can also allow two spAAV vectors containing the positive chain or negative chain of the target DNA gene to infect cells cultured in vitro separately, and the genome of the spAAV vector can be integrated into the cell genome to produce long-term and stable gene expression; and when the spAAV vectors containing the positive chain and negative chain of the target DNA gene respectively infect cells cultured in vitro (such as HEK 293 cells), the two can complement each other to produce double-stranded DNA molecules, which can express the target gene with higher efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is an exemplary schematic diagram of the DNA sequence structure of AAV2Flip ITR (including RBE and trs) provided by the present invention.

[0021] Figure 2 Schematic diagram of the packaging of the spAAV vector prepared by the insect cell baculovirus packaging system using the cis packaging element used in the examples of the present invention.

[0022] Figure 3 This is the SDS-PAGE electrophoresis diagram of the spAAV vector capsid protein produced by packaging with cis packaging elements in the embodiments provided by the present invention.

[0023] Figure 4 This is a neutral agarose gel electrophoresis diagram of the spAAV vector DNA produced by packaging the cis packaging element in the embodiments provided by the present invention.

[0024] Figure 5 Provided is a DNA denaturing alkaline agarose gel electrophoresis diagram of the capsid protein of the spAAV vector produced by packaging the cis packaging element provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0026] The meaning of scientific and technological terms in this application is consistent with the general understanding of those skilled in the art, unless otherwise specified. In this application, "one" or its combination with various quantifiers includes both singular and plural meanings, unless otherwise specified. In this application, for the same parameter or variable, when multiple numerical values, numerical ranges, or combinations thereof are given for description, it is equivalent to specifically revealing these numerical values, range ends, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it carries a modifier such as "about", covers an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this article, each "implementation method" refers to and covers the implementation methods of various methods and systems of this application equally. In this application, one or more technical features in any implementation method can be freely combined with one or more technical features in any one or more other implementation methods, and the implementation methods obtained thereby also belong to the content disclosed in this application.

[0027] Adeno-associated virus is a member of the Parvoviridae family. It has no envelope and an icosahedral symmetric structure. Currently, more than ten serotypes and more than a hundred variants of AAV have been discovered, among which AAV2 is the most thoroughly studied and widely used serotype.

[0028] The AAV genome consists of a linear single-stranded DNA of about 4.7 kb, with ITRs of about 145 bases at both ends. The approximately 125 bases near the end of the ITR contain a palindromic sequence, which can fold by itself through complementary base pairing, presenting a T-shaped hairpin structure. The ITR contains a Rep protein binding element (Rep binding site, RBE) and a terminal resolution site (trs), which can be recognized and bound by the Rep protein and produce a nick at the trs.

[0029] The AAV genome contains two open reading frames (ORFs) between the ITRs at both ends. These two ORFs encode rep and cap, respectively. The rep gene encodes four Rep proteins, Rep78, Rep68, Rep52, and Rep40, which play a role in the replication, integration, rescue, and packaging of the AAV virus. The cap gene encodes the capsid proteins VP1, VP2, and VP3 of the AAV virus, of which VP1 is necessary for the formation of infectious AAV, and VP3 is the main protein that makes up the AAV virus particles. The ratio of VP1, VP2, and VP3 in the AAV virus capsid is approximately 1:1:10.

[0030] Figure 1 The schematic diagram of the DNA sequence structure of AAV2 Flip ITR is shown, in which the ITR forms a T-shaped palindromic hairpin structure. Figure 1As shown in Figure 1, the first 125 bases of ITR can be divided into different segments, such as A', C', C, B', B, A, etc., where B and B', C and C' are reverse complementary to form a T-shaped palindromic hairpin structure, and A and A' form the handle of the hairpin. The D segment is also included after the A' segment. The positions of RBE and trs are shown in Figure 1. Figure 1 exemplified.

[0031] Although the ITR sequences are different in the AAV genomes of various serotypes, they can all form a T-shaped palindromic hairpin structure and have RBE and trs.

[0032] When AAV assembles to form virus particles, the positive and negative strand DNA genomes carrying wild-type ITRs are packaged into the AAV virus capsid with the same probability, thus producing the same number of positive and negative strand AAV virus particles. AAV virus particles infect and enter cells, uncapsidate in the cells, and release the AAV genome. After the ITRs at both ends of the AAV genome fold to form a T-shaped palindromic hairpin structure, the 3′ end is used as a primer to synthesize the second strand to form a double-stranded molecule, thereby initiating the expression of the genes carried in the AAV genome. The positive and negative strand DNA molecules of the AAV genome can also form double-stranded DNA to express genes through complementary pairing.

[0033] Based on this, the present invention provides a pre-packaging spAAV genome, wherein the 5′ end of the pre-packaging spAAV genome has a cis packaging element, and the 3′ end does not contain any cis packaging element sequence.

[0034] In the present invention, the term "monopolar" or "sp" refers to a single DNA polarity, that is, the spAAV vector produced by the pre-packaging spAAV genome packaging of the present invention has only one polarity, either a spAAV vector with positive-strand DNA or a spAAV vector with negative-strand DNA, and the two are not produced at the same time. The spAAV vector produced by the pre-packaging spAAV genome packaging described herein is also called a "monopolar rAAV vector" or "spAAV vector".

[0035] In the present invention, the term "recombinant adeno-associated virus vector" or "rAAV" refers to a non-wild-type adeno-associated virus particle that serves as a gene delivery vector and comprises a recombinant AAV genome packaged in a viral capsid.

[0036] In the present invention, the term "pre-packaging spAAV genome" refers to the recombinant AAV genome to be packaged and provided for subsequent packaging into capsid protein.

[0037] In the present invention, the term "open end" or "linear open end" refers to a 3' or 5' nucleotide sequence end that does not have any other structure (eg, hairpin structure) other than a linear straight chain.

[0038] In the present invention, the term "ITR or part thereof" refers to the whole or part of the DNA segment of the ITR, including the "ITR component". For example, in the case of Flip ITR (e.g. Figure 1 In the case of (as shown in the figure), the ITR components include but are not limited to: D region, trs site, RBE site, A′ region (part), C′ region, C region, B′ region, B region, A region, etc.

[0039] It is understood that the ITR sequences present at the ends of the AAV genome will typically be in the same or different segment orientations, e.g., Flip / Flop, Flop / Flip, Flip / Flip, or Flop / Flop. An exemplary structural orientation of a "Flip" ITR is as follows: Figure 1 An exemplary structural orientation of a "Flop" ITR is shown in Figure 1 As shown, the difference is that the positions of segment B′ and segment C′ are swapped, and the positions of segment B and segment C are swapped.

[0040] In the present invention, the term "hairpin structure" is also called "hairpin structure", which refers to a "hairpin" shape formed by the DNA molecule folding back on itself so that some bases in the folded region are close to each other and complementary to each other, such as a T-shaped hairpin structure, such as Figure 1 shown.

[0041] Different from the traditional rAAV genome before packaging, the spAAV vector formed by the spAAV genome before packaging in the present invention has only one polarity, that is, either a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time. In addition, the titer and cell transfection performance of the spAAV virus particles produced by packaging are comparable to those of traditional rAAV virus particles. The spAAV virus particles formed by packaging according to the present invention can advantageously deliver monopolar, single-stranded DNA of the same polarity with linear open ends (except for the use of ITR cis packaging elements). Since it is a monopolar, single-stranded DNA with only the same polarity, and there is no 3′ITR structure that can be used as a DNA replication primer, spAAV DNA can only exist in the form of single-stranded DNA in the cell, so that it can be used for gene integration, gene editing, and promoting efficient DNA pairing to express large genes and deliver long oligodeoxynucleotides, so that it can carry out small nucleic acid gene therapy and other work.

[0042] In some embodiments, the spAAV genome is single-stranded DNA.

[0043] In some embodiments, the 3′ end of the spAAV genome before packaging does not have an ITR or a portion thereof, and does not have other cis packaging elements.

[0044] In some embodiments, the 5′ end of the spAAV genome before packaging has the cis packaging element.

[0045] Since there is no ITR palindromic sequence at the 3′ end, the resulting spAAV vector cannot synthesize the second strand using the 3′ end as a primer and form a double-stranded DNA molecule after entering the host cell.

[0046] In some embodiments, the cis packaging element comprises trs and RBE.

[0047] In some embodiments, the cis packaging element comprises D, A' and C' segments derived from Flip ITR, for example, the corresponding structure is as follows Figure 1 as shown in .

[0048] In some embodiments, the cis packaging element comprises, among other things, D, A', and B' segments derived from Flop ITR.

[0049] In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises RBE and trs. In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises D, A′, and C′ segments derived from Flip ITR. In some exemplary embodiments, the spAAV genome before packaging comprises, in 5′ to 3′ order: a cis packaging element, an exogenous gene expression cassette, and / or optionally other exogenous DNA sequences, wherein the cis packaging element comprises D, A′, and B′ segments derived from Flop ITR.

[0050] In some exemplary embodiments, the cis packaging element includes any one of the DNA elements ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR; wherein the 5′ end of the spAAV genome before packaging has the cis packaging element; and the 3′ end of the spAAV genome before packaging does not have all or part of the DNA sequence of ITR and does not have other cis packaging elements.

[0051] In some exemplary embodiments, the ITR described in the present invention has the following nucleotide sequence (Flip ITR):

[0052] The sequence of Flip ITR is SEQ ID NO.1:

[0053]

[0054] In some exemplary embodiments, the ITR described in the present invention has the following nucleotide sequence (Flop ITR):

[0055] The sequence of Flop ITR is SEQ ID NO.2:

[0056]

[0057] In some exemplary embodiments, the nucleotide sequence of the ITR described herein has greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0058] In some exemplary embodiments, the nucleotide sequence of the ITR of the present invention is the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0059] In some exemplary embodiments, the nucleotide sequence of the RBE has the nucleotide sequence shown in SEQ ID NO: 3:

[0060] The sequence of RBE is SEQ ID NO. 3: gcgc gctc gctc gctc.

[0061] In some exemplary embodiments, the nucleotide sequence of RBE described in the present invention has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% with the nucleotide sequence shown in SEQ ID NO.3.

[0062] In some exemplary embodiments, the nucleotide sequence of trs in the present invention has the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.4: agttgg.

[0063] In some exemplary embodiments, the nucleotide sequence of trs in the present invention has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% with the nucleotide sequence shown in SEQ ID NO.4.

[0064] In some exemplary embodiments, the nucleotide sequence of ITRt described in the present invention has a sequence identity of greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, greater than 99% or 100% with the nucleotide sequence shown in SEQ ID NO.5.

[0065] Those skilled in the art will appreciate that the ITR, RBE, and trs sequences described above are for exemplary purposes only and are not intended to be limiting, and are described based on AAV2. Those skilled in the art will be able to determine the ITR sequences and RBE and trs sequences of each type (e.g., each serotype) of rAAV, and implement the present invention with appropriate adjustments without departing from the teachings of the present invention.

[0066] In some exemplary embodiments, the DtrsA′B′ described in the present invention has a nucleotide sequence shown in SEQ ID NO: 5:

[0067]

[0068] In some exemplary embodiments, the trsA′B′ has a nucleotide sequence as shown in SEQ ID NO: 6:

[0069]

[0070] In some exemplary embodiments, the trsA′ has a nucleotide sequence as shown in SEQ ID NO: 7:

[0071]

[0072] In some exemplary embodiments, the DttRR has a nucleotide sequence as shown in SEQ ID NO: 8:

[0073]

[0074] In some exemplary embodiments, the ttRR has a nucleotide sequence as shown in SEQ ID NO: 9:

[0075]

[0076] In some embodiments, the spAAV genome before packaging does not have a nucleotide sequence encoding a rep gene and / or a cap gene.

[0077] In some embodiments, the spAAV genome before packaging comprises an exogenous nucleic acid, for example, an exogenous gene expression cassette, between one end having a cis packaging element and the other end. The exogenous nucleic acid may have a nucleotide sequence for encoding an exogenous gene.

[0078] In some embodiments, the spAAV genome before packaging comprises an optional DNA sequence between one end having the cis packaging element and the other end; wherein the optional DNA sequence may be a nucleotide sequence that does not encode an exogenous gene, for example, a stuffer sequence.

[0079] In the present invention, the terms "exogenous gene", "exogenous nucleic acid", or "target gene" refer to genes or DNAs derived from organisms other than the organisms (e.g., individual organisms) of interest or research. The exogenous gene, exogenous nucleic acid, or target gene can be any exogenous gene or exogenous DNA that is expected to be expressed or produce biological functions in the recipient cells to which the spAAV virus particles are to be delivered.

[0080] In some embodiments, the spAAV genome comprises a positive-stranded single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0081] In some embodiments, the spAAV genome comprises a negative-strand single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0082] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a positive exogenous gene expression frame.

[0083] In some exemplary embodiments, the forward exogenous gene expression frame may include a promoter, an exogenous gene open reading frame, and a poly A sequence in the 5′-3′ direction.

[0084] In some exemplary embodiments, the positive exogenous gene expression frame may further include an enhancer and an intron.

[0085] In some exemplary embodiments, the enhancer and intron are located between the promoter and the open reading frame of the exogenous gene.

[0086] In some exemplary embodiments, the forward exogenous gene expression frame may include a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0087] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a reverse exogenous gene expression frame.

[0088] In some exemplary embodiments, the reverse exogenous gene expression frame may include, in the 5′-3′ direction, a reverse complementary sequence of a poly A sequence, a reverse complementary sequence of an exogenous gene open reading frame, and a reverse complementary sequence of a promoter.

[0089] In some exemplary embodiments, the reverse exogenous gene expression frame may further include an enhancer and an intron.

[0090] In some exemplary embodiments, the enhancer and intron are located between the reverse complementary sequence of the promoter and the reverse complementary sequence of the open reading frame of the foreign gene.

[0091] In some exemplary embodiments, the reverse exogenous gene expression cassette may include a reverse complementary sequence of a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0092] In some embodiments, the promoter may include, for example, a CMV promoter, a CAG promoter, or a cell-specific promoter, such as a GFAP promoter, a hAAT promoter, and the like.

[0093] In some embodiments, the pre-packaging spAAV genome can be produced by constructing a plasmid (such as a spAAV transgenic plasmid). The spAAV transgenic plasmid in the present invention can be selected from any plasmid suitable for producing the pre-packaging spAAV genome described herein. Suitable spAAV transgenic plasmids can be based on, for example but not limited to, pFastBacdual, pFastBac1 plasmids, etc.

[0094] The pre-packaging spAAV genome can subsequently be packaged into capsid protein through a spAAV vector packaging system to form a spAAV vector.

[0095] In some embodiments, the spAAV vector packaging system may include, for example, an insect cell baculovirus packaging system, a mammalian cell packaging system, and the like.

[0096] In another aspect, the present invention provides a spAAV vector comprising:

[0097] Capsid protein, and spAAV genome packaged by the capsid protein;

[0098] Wherein, the spAAV genome packaged by capsid protein is derived from the spAAV genome before packaging described in this article.

[0099] In the present invention, "spAAV vector" is also referred to as "spAAV virus particle", which is produced by packaging the pre-packaging spAAV genome into AAV capsid protein (for example, using a spAAV vector packaging system).

[0100] In some embodiments, examples of the spAAV vector packaging system may include, for example, an insect cell baculovirus packaging system, a mammalian cell packaging system, and the like.

[0101] In some embodiments, the spAAV genome packaged by capsid protein is a monopolar, single-stranded DNA.

[0102] In some embodiments, the spAAV genome packaged by capsid protein (or may also be referred to as "packaged spAAV genome") has linear open ends at both ends (except for the use of ITR cis packaging elements), one end does not have ITR or part thereof and does not have other cis packaging elements, and the other end has any cis packaging element.

[0103] In some embodiments, the 5′ end of the packaged spAAV genome has a cis packaging element-derived sequence.

[0104] In some embodiments, the packaged spAAV genome does not have a cis packaging element or a portion thereof at the 3′ end.

[0105] In some embodiments, the cis packaging element-derived sequence comprises RBE, trs.

[0106] In some embodiments, the cis packaging element-derived sequence comprises RBE and a portion of the trs sequence (the remaining trs portion after Rep cleavage).

[0107] In some embodiments, the packaged spAAV genome does not have a nucleotide sequence encoding a rep gene and / or a cap gene.

[0108] In some embodiments, the packaged spAAV genome comprises an exogenous nucleic acid, for example, an exogenous gene expression cassette, between one end having a cis packaging element-derived sequence and the other end. The exogenous nucleic acid may have a nucleotide sequence for encoding an exogenous gene.

[0109] In some embodiments, the nucleotide sequence encoding the exogenous gene is a monopolar, single-stranded DNA sequence.

[0110] In some embodiments, the packaged spAAV genome comprises an optional DNA sequence between one end having a cis packaging element-derived sequence and the other end; the optional DNA sequence may be a nucleotide sequence that does not encode an exogenous gene, for example, a stuffer sequence.

[0111] In some embodiments, the packaged spAAV genome comprises a positive-strand single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0112] In some embodiments, the packaged spAAV genome comprises a negative-strand single-stranded DNA sequence of an exogenous nucleic acid (eg, an exogenous gene expression cassette).

[0113] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a positive exogenous gene expression frame.

[0114] In some exemplary embodiments, the positive exogenous gene expression frame may include a promoter, an exogenous gene open reading frame, and a poly A sequence in the 5′-3′ direction. In some exemplary embodiments, the positive exogenous gene expression frame may also include an enhancer and an intron.

[0115] In some exemplary embodiments, the enhancer and intron are located between the promoter and the open reading frame of the exogenous gene.

[0116] In some exemplary embodiments, the forward exogenous gene expression frame may include a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0117] In some embodiments, the nucleotide sequence encoding the exogenous gene includes a reverse exogenous gene expression frame.

[0118] In some exemplary embodiments, the reverse exogenous gene expression frame may include, in the 5′-3′ direction, a reverse complementary sequence of a poly A sequence, a reverse complementary sequence of an exogenous gene open reading frame, and a reverse complementary sequence of a promoter.

[0119] In some exemplary embodiments, the reverse exogenous gene expression frame may further include an enhancer and an intron.

[0120] In some exemplary embodiments, the enhancer and intron are located between the reverse complementary sequence of the promoter and the reverse complementary sequence of the open reading frame of the foreign gene.

[0121] In some exemplary embodiments, the reverse exogenous gene expression cassette may include a reverse complementary sequence of a DNA sequence for gene editing or gene regulation in the 5′-3′ direction.

[0122] In some embodiments, the promoter may include, for example, a CMV promoter, a CAG promoter, or a cell-specific promoter, such as a GFAP promoter, a hAAT promoter, and the like.

[0123] In some embodiments, the capsid protein is an AAV capsid protein. In some embodiments, the capsid protein can be a capsid protein of various AAVs selected from AAV1-AAV12 (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12).

[0124] In some embodiments, the capsid protein is an AAV capsid protein variant, such as a tyrosine single amino acid / multiple amino acid variant, a tyrosine, serine, or threonine variant, or a multi-serotype chimera such as AAV-DJ, or a polypeptide embedded variant, etc.

[0125] In some embodiments, the capsid protein can be produced from a plasmid expressing the AAV capsid protein Cap by utilizing a spAAV vector packaging system.

[0126] In some embodiments, the spAAV vector can be packaged and produced by a spAAV transgenic plasmid (producing the spAAV genome before packaging) and a plasmid expressing the AAV capsid protein Cap and the replication protein Rep (producing Cap and Rep proteins) using a spAAV vector packaging system.

[0127] In some embodiments, the AAV Cap encoding gene and the AAV Rep encoding gene can be on the same plasmid or on two different plasmids.

[0128] In some embodiments, the AAV Cap and / or AAV Rep expression plasmid may include a Cap and / or Rep gene expression cassette and desired expression elements, as well as an intron sequence for enhancing expression, etc. There is no particular limitation on the plasmid used to express AAV Cap and / or Rep, and those skilled in the art can routinely apply AAV Cap and / or Rep expression plasmids, see, for example, Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 12427305.

[0129] On the other hand, the present invention also provides a spAAV transgenic plasmid, which is used to produce the spAAV genome before packaging described herein.

[0130] The spAAV transgenic plasmid described in the present invention can be selected from any plasmid suitable for producing the spAAV genome before packaging described herein, wherein the suitable spAAV transgenic plasmid can be based on, for example but not limited to, pFastBacdual plasmid, pFastBac1 plasmid, etc.

[0131] In some exemplary embodiments, the spAAV transgenic plasmid can be designed as a positive-stranded single-stranded DNA sequence encoding an exogenous gene, which may include, in 5′ to 3′ order: a cis packaging element (including RBE and trs), a promoter (such as CMV), an exogenous gene open reading frame, and poly A.

[0132] In some exemplary embodiments, the spAAV transgenic plasmid may further include a stuffer sequence.

[0133] In some embodiments, the stuffer sequence can be present, for example, upstream of the promoter and / or downstream of the poly A.

[0134] In some exemplary embodiments, the spAAV transgenic plasmid can be designed as a negative-stranded single-stranded DNA sequence encoding an exogenous gene, which may include, in 5' to 3' order: cis packaging element (including RBE and trs), poly A, exogenous gene open reading frame ', promoter ' (such as CMV '). In some exemplary embodiments, the spAAV transgenic plasmid may also include a stuffer sequence. In some specific embodiments, the stuffer sequence may be present, for example, downstream of the promoter 'and / or upstream of the poly A '.

[0135] In the present invention, the term "stuffing sequence" refers to an auxiliary DNA that is added to fill in the length of DNA to reach the packaging length of AAV when the length of the target DNA is less than 4.7 kb. In some embodiments, the stuffing sequence can have different lengths as long as the final DNA reaches a length of, for example, about 4.7 kb.

[0136] The spAAV transgenic plasmid can be packaged together with the Rep and Cap gene expression plasmids through the spAAV vector packaging system to produce the spAAV vector described in this article.

[0137] On the other hand, the present invention also provides a method for packaging spAAV vectors, which comprises: transforming the spAAV transgenic plasmid and the Cap and Rep expression plasmids into Escherichia coli competent cells respectively; picking out white colonies after at least one round of blue-white screening, amplifying and extracting recombinant bacmids; transfecting insect cells with the recombinant bacmids to produce recombinant baculoviruses; and extracting the recombinant baculoviruses and infecting insect cells with the recombinant baculoviruses to obtain spAAV vectors.

[0138] In some embodiments, the E. coli competent cells are E. coli DH10Bac competent cells.

[0139] In some embodiments, the spAAV vector packaging system is an insect cell baculovirus packaging system.

[0140] In some embodiments, after at least one round of blue-white screening, white colonies are picked, amplified and recombinant bacmids are extracted.

[0141] In some embodiments, after two or more rounds of blue-white screening, white colonies are picked, amplified, and recombinant bacmids are extracted.

[0142] In some embodiments, the recombinant bacmid is transfected into insect cells (eg, insect cells Sf9) to produce recombinant baculovirus (eg, P3 generation recombinant baculovirus).

[0143] In some embodiments, recombinant baculovirus (eg, two or three recombinant baculoviruses of generation P3) is infected (eg, co-infected) with insect cells (eg, Sf9 insect cells) to package and obtain a spAAV vector.

[0144] like Figure 2 As shown, a schematic diagram of packaging a spAAV vector according to an exemplary embodiment of the present invention is given. In a specific embodiment, when the spAAV vector is packaged, the Rep protein recognizes the Rep binding site (RBE) on the recombinant baculovirus DNA, binds to the site, recognizes the terminal melting site (trs), opens the phosphodiester bond between the bases tt, and covalently binds to the 5′ end, introducing the DNA into the AAV capsid, and the length of the DNA packaged into the capsid is about 4.7 kb.

[0145] For methods of packaging rAAV vectors using the insect cell baculovirus packaging system, see, for example, Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 1242730.

[0146] On the other hand, the present invention also provides a method for delivering exogenous nucleic acid to a cell, comprising contacting the cell with one or more spAAV vectors described herein, wherein the genome of one or more of the spAAV vectors comprises a gene expression frame of the exogenous nucleic acid and / or optional other DNA sequences.

[0147] In some embodiments, the cell can be a eukaryotic cell.

[0148] In some embodiments, the cell may be an animal cell.

[0149] In some embodiments, the cell may be a vertebrate cell.

[0150] In some embodiments, the cell can be a mammalian cell, such as a human cell.

[0151] In some embodiments, the exogenous nucleic acid comprises the nucleic acid sequence of an exogenous gene.

[0152] In some embodiments, the exogenous nucleic acid comprises a nucleic acid sequence that is not an exogenous gene.

[0153] In some embodiments, the genome of the spAAV vector comprises a positive-stranded single-stranded DNA sequence or a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0154] In some embodiments, one or more of the spAAV vectors include: a spAAV vector containing only the positive-strand single-stranded DNA sequence of the exogenous nucleic acid and / or a spAAV vector containing only the negative-strand single-stranded DNA sequence of the exogenous nucleic acid.

[0155] In some embodiments, one or more of the spAAV vectors include two or more spAAV vectors, which include at least a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0156] In some embodiments, the cell is contacted with a spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene.

[0157] In some embodiments, the cell is contacted with a spAAV vector having a negative-sense single-stranded DNA sequence of the exogenous gene.

[0158] In a preferred embodiment, the cells are contacted with a first spAAV vector having a positive-strand single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-strand single-stranded DNA sequence of the exogenous gene.

[0159] In some embodiments, the cells are contacted simultaneously with a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene.

[0160] In some embodiments, the cells are sequentially (e.g., sequentially within 24 hours) contacted with a first spAAV vector having a positive-stranded single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene, or vice versa. It should be understood that in the present invention, "a first spAAV vector" and "a second spAAV vector" are only used to distinguish between the two and are not intended to limit the order of their use.

[0161] When cells are infected with a spAAV vector having a positive-strand single-stranded DNA sequence of the exogenous gene, or a spAAV vector having a negative-strand single-stranded DNA sequence of the exogenous gene, the exogenous gene carried by the vector has a greater probability (for example, 20%) of being integrated into the cell genome, resulting in long-term and stable exogenous gene expression.

[0162] By using a spAAV vector having a positive-strand single-stranded DNA sequence of the exogenous gene and a spAAV vector having a negative-stranded single-stranded DNA sequence of the exogenous gene in combination, it is beneficial for the spAAV genome containing the positive and negative strands to quickly complement each other in the nucleus of the host cell to form a double-stranded molecule, thereby initiating gene expression.

[0163] In another aspect, the present invention also provides an isolated host cell comprising the spAAV vector described herein.

[0164] In some embodiments, the host cell can be a eukaryotic cell.

[0165] In some embodiments, the host cell may be an animal cell.

[0166] In some embodiments, the host cell may be a vertebrate cell. In some embodiments, the host cell may be a mammalian cell.

[0167] In some embodiments, the host cell can be a human cell.

[0168] In some embodiments, the host cell is obtained by contacting a eukaryotic cell (eg, an animal cell, eg, a mammalian cell, eg, a human cell) with the spAAV vector of the present invention.

[0169] On the other hand, the spAAV vector of the present invention is also provided, which is used for gene expression. Also provided is the use of the spAAV described herein in preparing a product for gene expression.

[0170] In some embodiments, the subject of gene expression is an animal, specifically a vertebrate, more specifically a mammal, and more specifically a human.

[0171] On the other hand, the spAAV vector of the present invention is also provided, which is used for gene therapy. Specifically, the present invention provides the use of spAAV in preparing products for gene therapy.

[0172] In some embodiments, the subject of gene therapy is an animal, particularly a vertebrate, more particularly a mammal, and even more particularly a human.

[0173] On the other hand, the spAAV vector of the present invention is also provided, which is used for gene editing. Also provided is the use of the spAAV described herein in preparing a product for gene editing.

[0174] In some embodiments, the subject of gene editing is an animal, specifically a vertebrate, more specifically a mammal, and more specifically a human.

[0175] On the other hand, the spAAV vector described herein is also provided, which is used for gene regulation. Also provided is the use of the spAAV described in the present invention in preparing a product for gene regulation.

[0176] In some embodiments, the subject of gene regulation is an animal, specifically a vertebrate, more specifically a mammal, and more specifically a human.

[0177] The technical solution of the present invention is further described in detail below through specific embodiments.

[0178] Example 1

[0179] This example constructs a plasmid vector and spAAV vector insect cell packaging system for spAAV vector packaging. The specific steps include:

[0180] First, the construction of plasmid vectors for spAAV vector packaging;

[0181] Second, the preparation of recombinant Bacmid;

[0182] Third, preparation of baculovirus;

[0183] Fourth, spAAV vector packaging;

[0184] Finally, the spAAV vector is detected and identified; this step includes first purifying using iodixanol density gradient ultracentrifugation, then detecting the titer of the spAAV vector by fluorescent quantitative PCR, analyzing the spAAV vector capsid protein by SDS-PAGE electrophoresis, identifying the spAAV vector genome, DNA neutral agarose gel electrophoresis analysis, DNA alkaline agarose gel electrophoresis analysis, annealing of the spAAV genome containing the positive and negative strands of the target gene DNA to identify the monopolar genome, S1 enzyme digestion to identify the spAAV vector single-stranded DNA genome, and gene expression in spAAV vector-infected HEK293 cells.

[0185] The above steps are described in detail below.

[0186] First, the construction of plasmid vectors for spAAV vector packaging

[0187] In this embodiment, the rAAV transgenic plasmid containing DtrsA′B′ related sequences only at one end of the exogenous gene encoding expression frame is also referred to as a “monopolar” plasmid, wherein the main plasmid involved is the spAAV DNA genomic plasmid.

[0188] The construction method specifically includes:

[0189] 1.spAAV-EGFP(+)DNA genome plasmid

[0190] The plasmid DNA contains D sequence, trs sequence, A′B′ sequence, 1.3kb stuff1 stuffing sequence, forward EGFP gene expression frame (CMV-EGFP-polyA) sequence, 1.3kb stuff2 stuffing sequence, and the entire packaging frame is 5.0kb.

[0191] The construct used pFastBacdual-ITR-EGFP as the vector.

[0192] The DtrsA′B′ sequence (5′ to 3′ direction) is SEQ ID NO: 5, specifically:

[0193]

[0194] The sequence of the EGFP gene expression frame is promoter CMV, enhancer, intron, EGFP open reading frame, polyA sequence;

[0195] The first step was to construct pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff.

[0196] DtrsA′B′ is obtained by denaturing and annealing (annealing) artificially synthesized primer 1 and primer 2.

[0197] Primer 1 and Primer 2 were both synthesized by Sangon Biotech (Shanghai) Co., Ltd.; wherein the sequence of Primer 1 is SEQ ID NO.6:

[0198]

[0199] The sequence of primer 2 is SEQ ID NO.7:

[0200]

[0201] The denaturation and renaturation (annealing) steps include: 5 μL of primer 1 and primer 2 oligonucleotides (concentration of 200 μM), 4 μL of 5× annealing buffer, and supplemented with deionized water without DNase and RNase to a final volume of 20 μL. The reaction was carried out at 95°C for 4 minutes in a PCR instrument for annealing, and then the reaction tube was taken out and placed on the laboratory bench, and cooled to room temperature for 5-10 minutes to form oligo-dsDNA with KpnI / MluI sticky ends, which was inserted into the KpnI / MluI double-enzyme-digested pFastBacdual-ITR-EGFP vector to obtain the vector pFastBacdual-DtrsA′B′-CMV-EGFP-polyA.

[0202] RHstuff was obtained by denaturation and renaturation (annealing) of artificially synthesized primers 3 and 4, both of which were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0203] The sequence of primer 3 is SEQ ID NO. 8: 5′-GACCGATAATCTCTAGATTGAGGAACAA-3′;

[0204] The sequence of primer 4 is SEQ ID NO. 9: 5′-AGCTTTGTTCCTCAATCTAGAGATTATCG-3′.

[0205] Primer 3 and primer 4 used the same denaturation and renaturation steps as above to form oligo-dsDNA with RsrII / HindIII sticky ends, which were inserted into the RsrII / HindIII double-digested pFastBacdual-DtrsA′B′-CMV-EGFP-polyA vector to obtain the pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector.

[0206] The second step was to construct pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGFP-polyA-1.3kbstuff2.

[0207] First, 1.3 kb stuff1 was fused with a CMV fragment by PCR and cloned to obtain a 1.3 kb stuff1-CMV fragment. The primers included primer 5, primer 6, primer 7 and primer 8, all of which were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0208] The sequence of primer 5 is SEQ ID NO.10:

[0209]

[0210] The sequence of primer 6 is SEQ ID NO.11:

[0211]

[0212] The sequence of primer 7 is SEQ ID NO.12:

[0213]

[0214] The sequence of primer 8 is SEQ ID NO.13:

[0215]

[0216] PCR reaction system: KOD-Plus-Neo 2.5μL, 10x PCR Buffer 5μL, 25mM MgSO45μL, 25mMMgSO4 5μL, DMSO 5μL, primer 5 and primer 6 (10uM) 0.6μL each, gene template saCas91-50ng, add H2O to make up to 50μL.

[0217] The PCR reaction program was: 95°C for 4 min; 95°C for 10 s, 56°C for 30 s, 72°C for 3 min, for 25 cycles.

[0218] A 1.3 kb stuff1 fragment was obtained by PCR amplification; a CMV fragment was obtained by amplification using primers 7 and 8 and template pFastBacdual-ITR-EGFP; a 1.3 kb stuff1-CMV fragment was obtained by fusion PCR amplification using primers 5 and 8 and templates 1.3 kb stuff1 and CMV.

[0219] A 1.3 kb stuff1-CMV fragment was obtained by fusion PCR amplification and inserted into the pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector by MluI / EcoRI double restriction digestion to obtain the pFastBacdual-DtrsA′B′-1.3 kb stuff1-CMV-EGFP-polyA-RHstuff vector.

[0220] The 1.3 kb stuff2 fragment was obtained by PCR cloning. The primers included primer 9 and primer 10, both of which were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0221] The sequence of primer 9, SEQ ID NO: 14, is:

[0222]

[0223] The sequence of primer 10, SEQ ID NO: 15, is:

[0224]

[0225] The PCR reaction system included KOD-Plus-Neo 2.5 μL, 10x PCR Buffer 5 μL, 25 mM MgSO4 5 μL, 25 mM MgSO4 5 μL, DMSO 5 μL, 0.6 μL each of primers 9 and 10 (10 μM), gene template saCas91-50 ng, and H2O was added to make up to 50 μL.

[0226] The PCR reaction program was: 95°C for 4 min; 95°C for 10 s, 56°C for 30 s, 72°C for 1.5 min, for 25 cycles.

[0227] The 1.3kb stuff2 fragment obtained by PCR amplification was inserted into the pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGF-polyA-RHstuff vector by single restriction digestion with BstEII to obtain the pFastBacdual-DtrsA′B′-1.3kbstuff1-CMV-EGFP-polyA-1.3kb stuff2 vector.

[0228] So far, we have obtained

[0229] pFastBacdual-DtrsA′B′-1.3kb stuffl-CMV-EGF-polyA-1.3kb stuff2 (spAAV-CMV-EGFP(+)) vector, that is, spAAV-CMV-EGFP(+) genome packaging plasmid.

[0230] The construction of the EGFP expression frame in this embodiment is a prior art, and specific references can be made to the literature: Li Taiming et al., Preparation of AAV-ITR Gene Expression Microvectors in Insect Cells, Chinese Journal of Biotechnology, 2015, 31(8), page 1232, "Construction of pFastBacdual-ITR-EGFP Plasmid" in method 1.2.1.

[0231] 2. pAAV-EGFP(-)DNA genome plasmid

[0232] The plasmid DNA contains D sequence, trs sequence, A'B' sequence, and reverse EGFP gene expression frame sequence from 5' to 3'

[0233] The DtrsA′B′ sequence is SEQ ID NO: 5, including

[0234]

[0235] The reverse EGFP gene expression frame sequence, namely the polyA sequence, the reverse complementary sequence of the EGFP open reading frame, introns, enhancers, promoters, and downstream Rep recognition and cleavage sequences.

[0236] The reverse EGFP expression cassette was inserted into the

[0237] pFastBacdual-DtrsA′B′-1.3kb stuff1-CMV-EGF-polyA-1.3kb stuff2 was used as a template to clone the 1.3kb stuff2′-polyA′-EGFP′-CMV′-1.3kb stuff1′ fragment by PCR, and the target fragment was obtained after PCR amplification using primers 11 and 12. Among them, primers 11 and 12 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0238] The sequence of primer 11, SEQ ID NO: 16, includes:

[0239]

[0240] The sequence of primer 12, SEQ ID NO: 17, includes:

[0241]

[0242] PCR fragments were inserted into the

[0243] pFastBacdual-DtrsA′B′-CMV-EGFP-polyA-RHstuff vector was obtained.

[0244] pFastBacdual-DtrsA′B′-1.3kb stuff2′-polyA′-EGFP′-CMV′-1.3kb stuff1′(spAAV-EGFP(-)) genome packaging plasmid.

[0245] Second, the preparation of recombinant Bacmid.

[0246] Specifically, the spAAV vector packaging plasmid prepared by the above steps and the three recombinant plasmids are used to prepare recombinant Bacmids respectively, and the recombinant method is as follows:

[0247] 1. Slowly thaw 100ul DH10Bac competent cell on ice.

[0248] 2. Add 500 ng of plasmid DNA and mix gently.

[0249] 3. Place on ice for 30 minutes, heat shock at 42°C for 90 seconds, and immediately place on ice for 2 minutes.

[0250] 4. Add 900 μL LB medium and culture on a shaking platform at 37°C and 225 rpm for 2-3 hours.

[0251] 5. Add 40 μL of 2% (20 mg / mL) galactoside X-gal and 20 μL of 20% (200 mg / mL) isopropyl-β-d-thiogalactoside IPTG to the center of a pre-made 90 mm agar plate containing 50 μg / mL kanamycin Kan, 7 μg / mL gentamicin Gen, and 10 μg / mL tetracycline Tet.

[0252] Use a sterile applicator to spread it over the entire surface of the culture plate and incubate at room temperature until all the liquid disappears.

[0253] 6 Dilute the cells 10-fold in LB medium (10 -1 , 10 -2 ), 100 μL of each gradient was applied to LB plate.

[0254] 7. Place at 37°C for 48 hours, pick two white clones, dip them onto a new LB agar plate (same resistance as above), streak them, and incubate at 37°C overnight.

[0255] 8. Pick two single plaques for PCR identification; PCR identification of Bacmid, the primers used are target gene F / R, PUC M13F / R.

[0256] 9. Take out the correctly identified Bacmid plaque, inoculate it into 10mL LB (Kan+, Gen+, Tet+), shake the bacteria for 12h, and use the OMEGA kit to extract and separate the recombinant bacmid DNA. The experimental method refers to the instruction manual of the OMEGA kit. After measuring the bacmid concentration, divide it into aliquots and freeze it at -20℃. The recombinant Bacmid is now obtained.

[0257] Third, preparation of baculovirus.

[0258] The specific steps of preparing baculovirus include:

[0259] 1. Prepare transfection reagent

[0260] 1×HBS 200mL preparation

[0261] Hepes 0.954g

[0262] NaCl 1.754g

[0263] Sterile ddH2O 150mL

[0264] Adjust pH to 7.4 with 1M NaOH, make up to 200 mL, filter aseptically in a safety cabinet, and store at 4°C

[0265] PEI 20mL preparation method includes

[0266] PEI 0.043g

[0267] 1 mL of anhydrous ethanol

[0268] After fully dissolved, add 1×HBS to 20 mL, freeze and thaw three times (freeze at -20°C, thaw at room temperature), and store at -20°C

[0269] 2. Cell Plating

[0270] Plating: Take a 6-well plate, aspirate the suspended culture cells and count them, so that the density of the plated cells is 2x10 6 cells / mL, 2mL per well, and the cell survival rate was above 95%.

[0271] 3. Transfection (amount per well)

[0272] Solution A: PEI: Add 6 μL PEI and 94 μL 1 × HBS, mix well, and let stand for 4 minutes.

[0273] Solution B: Take 3 μg of bacmid DNA (inactivate the bacmid at 65°C for 30 minutes in advance), add 1×HBS to make the final volume 100 μL, and mix gently.

[0274] Take 100 μL of solution A and add it to solution B, mix well, and incubate at room temperature for 30 minutes. Add it to the wells with cells and culture for 96 hours.

[0275] 4. Virus Amplification

[0276] 1) Separation of P1

[0277] After confirming that the cells were in the late infection stage (96 h), 2 mL of virus-containing culture medium was collected from each well into a sterile 15 mL centrifuge tube and centrifuged at 500 g for 5 min to remove cell debris.

[0278] Take the supernatant into a sterile EP tube and store it at 4℃ away from light. If you want to store it for a long time, freeze it in aliquots at -80℃.

[0279] 2) Amplify the virus to obtain P2

[0280] Take 8 mL of suspension cultured cells with a multiplicity of infection (MOI) of 0.1 and a density of 2×10 6cells / mL, the required volume of P1 is calculated to be 1.6 mL, culture at 27°C for 72 h, collect the suspended cultured cells in a sterile 15 mL centrifuge tube, and centrifuge at 500 g for 5 minutes. Aliquot the supernatant into a sterile EP tube. The viral supernatant is P2, which is stored at 4°C in the dark, or can be frozen at -80°C to extend the storage time.

[0281] 3) Amplify the virus to obtain P3

[0282] P3 can be obtained by amplification according to the above method, with an MOI of 0.1 and a density of 2×10 6 cells / mL, 200 μL P2 storage solution was added to 10 mL of suspended culture cells, and P3 was collected after culturing for 72 h.

[0283] The P1 virus titer is usually around 1×10 6 -1×10 7 The P2 titer is between 1×10 7 ~1×10 8 between.

[0284] 4) Virus packaging

[0285] Take 100 mL of suspension cultured cells at an MOI of 1 and a density of 5 × 10 6 cells / mL, add 5 mL each of P3 generation Rep, Cap, and target gene, culture for 72 hours, and collect the cells.

[0286] Fourth, preparation of spAAV vector.

[0287] The spAAV vector packaging system of the present invention can be an insect cell baculovirus packaging system or a mammalian cell packaging system. The insect cell is an Sf9 cell.

[0288] This embodiment takes the insect cell baculovirus packaging system as an example for description, and the specific steps include:

[0289] First, one or two recombinant plasmids expressing AAV replication protein Rep and AAV capsid protein Cap (Rep and Cap genes are on one or two different plasmids) and another plasmid containing spAAV DNA genome are transformed into E. coli DH10Bac competent cells respectively, and after two rounds of blue-white screening, the colonies containing recombinant bacmids are white and the colonies without recombination are blue. The white colonies are selected for amplification and the recombinant bacmids are extracted.

[0290] Secondly, the above two or three recombinant bacmids were transfected into insect cells Sf9 using an insect cell transfection reagent. After 4 to 5 days, the cell supernatant was collected to obtain the P1 generation insect cell recombinant baculovirus. The P1 generation recombinant baculovirus was amplified by infecting Sf9 insect cells twice to obtain the P3 generation recombinant baculovirus. The titer of the P3 generation baculovirus was determined by the plaque method, and the virus titer (pfu / mL) = 1 / dilution multiple × number of plaques × 1 / inoculation volume per well.

[0291] Finally, two or three recombinant baculoviruses of the P3 generation are co-infected with sf9 insect cells to package and obtain spAAV vector virus particles. For the operation, please refer to the literature: Urabe M, Ding C, Kotin RM. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther. 2002 Nov 1; 13(16): 1935-43. doi: 10.1089 / 10430340260355347. PMID: 12427305.

[0292] See also Figure 2 , which is a schematic diagram of the packaging of the spAAV vector prepared by the insect cell baculovirus packaging system in this embodiment. In the figure, when the spAAV vector is packaged, the Rep protein recognizes the Rep binding site (RBE) on the recombinant baculovirus DNA, binds to the site, recognizes the terminal melting site (trs), opens the phosphodiester bond between the bases tt, and covalently binds to the 5′ end, introducing the DNA into the AAV capsid, and the length of the DNA packaged into the capsid is about 4.7 kb.

[0293] Finally, detection and identification analysis

[0294] 1. Virus Purification

[0295] The purification was performed by iodixanol density gradient ultracentrifugation, and the specific steps included:

[0296] 1. Obtain cell lysate

[0297] 1) 72 h after co-transfection, cells were collected by centrifugation at 1000 g for 5 min;

[0298] 2) Discard the supernatant and resuspend the cells in 10 mL Lysis Buffer;

[0299] Lysis Buffer(1L):NaCl 8.766g

[0300] Tris 6.055g

[0301] DH2O 950ml

[0302] 5M HCl adjust pH to 8.5, and make up to 1L

[0303] Sterile filtration in a safety cabinet and stored at 4°C

[0304] 3) Freeze in liquid nitrogen and thaw at 37°C, repeat 3 to 4 times until clear;

[0305] 4) Centrifuge at 4°C, 5000g for 30 minutes to collect the supernatant; resuspend the precipitate in 4-5mL PBS and centrifuge again to collect the supernatant;

[0306] 5) Add DNase (10 μL / mL) and RNase (1 μL / mL) and digest in a 37°C water bath for 1 h for purification;

[0307] 2. Virus Purification by Iodixanol Density Gradient Ultracentrifugation

[0308] 1) Dilute 60% iodixanol to 15%, 25%, 40% and 58% with PBS-MK (1×PBS, 1mM MgCl2, 2.5mM KCl), and add solid NaCl to the 15% phase to a final concentration of 1M; 2) Add 0.5% phenol red solution to 1.5μL / mL in the 15% and 25% phases, and add 0.5% phenol red solution to 0.5μl / mL in the 58% phase;

[0309] 3) Use a 1.27*120mm flat-blade spinal puncture needle to add 8mL 15%, 6mL 25%, 8mL 40% and 5mL 58% iodixanol diluents from the bottom of a 39mlL quick-seal tube in sequence, avoiding the formation of bubbles;

[0310] 4) Carefully overlay the cell lysate on the iodixanol density gradient solution and fill the tube (to the level of the tube mouth line). If necessary, fill the tube with Lysis Buffer to make up the volume, balance it, and centrifuge it at 69,000 rpm, 18°C ​​for 1.5 h (BECKMANCOULTER centrifuge 70Ti rotor);

[0311] 5) Pierce the quick-seal tube from the bottom, discard the first 4 mL (corresponding to the 58% phase), and collect 6 mL of the solution in the tube;

[0312] 6) Ultrafiltration tube (Amicon Uitra-4 Centrifugai Filters, Ultracel-100k), 3000g, centrifugation for 5 minutes; repeated washing with PBS (1×PBS, Solebol, supplemented with 5M NaCl to a final concentration of 350mM) until the residual concentration of iodixanol is <0.1%, and the final density per 100mL packaging volume is compressed to about 200μL;

[0313] 7) Add glycerol to a final concentration of 5%, sterile filter, and store in aliquots at -80°C;

[0314] 2. Fluorescence Quantitative Detection

[0315] The method for quantitatively detecting the titer of spAAV vector by fluorescent PCR (RT-PCR method) specifically includes:

[0316] 1. Dilute the plasmid ITR-EGFP 10 times, then perform gradient dilution, and dilute 5 gradients to make a standard curve

[0317] 2. Dilute the virus 10 times, then perform gradient dilution, dilute 4 gradients

[0318] 3. Buffer preparation (protect from light):

[0319] 2xSuperpreMix Plus(SYBR Green) 10μL

[0320] Upstream primer, 5'-TCCGCGTTACATAACTTACGG-3' (SEQ ID NO: 22; synthesized by Shanghai Sangon Biotechnology Co., Ltd.), 0.3 μL.

[0321] Downstream primer, 5'-GGGCGTACTTGGCATATGAT-3' (SEQ ID NO: 23; synthesized by Shanghai Biotech Co., Ltd.), 0.3 μL.

[0322] DD H2O 4.4μL

[0323] 4. Sample addition, 20μL system: 5μL template + 15μL Buffer, two replicate wells for each gradient

[0324] 5.RT-PCR (Roche LightCycler) procedure:

[0325]

[0326] 3. Analysis of spAAV vector capsid protein by SDS-PAGE electrophoresis

[0327] 1. Glue making:

[0328] 1) Separation gel (10%): Add the following materials (9.093 mL) to the beaker for separation gel: 3.69 mL of double distilled water, 2.97 mL of 30% acrylamide gel solution (acrylamide: methylene bisacrylamide = 29:1), 2.25 mL of 2M Tris, pH 8.8, 90 μL of 10% ammonium persulfate, 90 μL of 10% SDS, and 3 μL of TEMED. Mix well and drop the separation gel between two glass plates with a dropper until the liquid level reaches 1 cm below the lower edge of the comb; slowly add 75% ethanol with a dropper, let it stand, and after the separation gel is polymerized, pour off the 75% ethanol layer.

[0329] 2) Concentrating gel (5%): Add the following materials (2.357 mL) to the beaker for preparing the concentrated gel: 1.83 mL of double distilled water, 0.39 mL of 30% acrylamide gel solution (acrylamide: methylene bisacrylamide = 29:1), 0.5 M Tris, pH 6.8 0.75 mL, 30 μL of 10% ammonium persulfate, 30 μL of 10% SDS, and 2 μL of TEMED. Mix well and immediately use a dropper to add the concentrated gel to cover the separation gel between the two glass plates until it is full. Gently insert a comb and let it stand until it solidifies to make a gel plate.

[0330] 2. Sample processing:

[0331] Virus sample: 5X loading buffer = 2:1 ratio, mix well, boil in a boiling water bath for 10 minutes, centrifuge immediately after boiling, and take all samples for electrophoresis.

[0332] 3. Electrophoresis

[0333] 1) Install the gel plate made of two glass plates vertically on the power supply rack in the electrophoresis tank, so that the concave edge of the gel plate is close to the power supply rack.

[0334] 2) Fix the gel plate and power supply rack in the power supply slot as required, add electrophoresis buffer as required, and gently remove the comb in the gel plate.

[0335] 3) Take the treated sample solution, use a microinjector to draw 15 μL of the sample solution and slowly add it to the notch in the gel plate (the sample entry point).

[0336] 4) During electrophoresis, control the voltage to 90V for the concentrated gel and 120V for the separation gel. Stop electrophoresis when the bromophenol blue strip in the gel is observed to be 1 cm from the bottom.

[0337] 4. Staining. Use a blade or a thin plate to gently pry open the two glass plates outside the gel plate, and use a blade to cut the separation gel along the junction of the separation gel and the concentrated gel on the gel. Then carefully move the separation gel into the staining container, add 100mL of staining solution, cover it, and stain for 1-3 hours.

[0338] 5. Decolorization. Pour away the staining solution. Rinse the stained gel several times with water, put it into clean water until the water just covers the gel, put it into a microwave oven, heat it on high heat for 2 minutes, take it out and shake it slowly, and repeat this operation until the protein bands can be clearly seen.

[0339] Figure 3 The capsid protein SDS-PAGE electrophoresis results of spAAV vectors packaged with cis packaging elements are shown. Lane MW: protein molecular weight standard; Lane 1: spAAV vector packaged with ITR cis element; Lane 2: spAAV vector packaged with DtrsA′B′ cis element; Lane 3: spAAV vector packaged with trsA′B′ cis element; Lane 4: spAAV vector packaged with trsA′ cis element; Lane 5: spAAV vector packaged with DttRR cis element; Lane 6: spAAV vector packaged with ttRR cis element.

[0340] As can be seen from the figure, there is no difference in the composition of the spAAV vector capsid protein produced by packaging various cis packaging elements and the ordinary rAAV capsid protein. They are all composed of three proteins: Vp1, Vp2, and Vp3, and the composition of the three proteins is basically 1:1:10.

[0341] 4. spAAV vector genome identification

[0342] First, DNA neutral agarose gel electrophoresis analysis is performed, and the specific steps include:

[0343] 1. Prepare agarose solution by adding an accurate amount of agarose powder and a quantitative amount of 1X TAE electrophoresis buffer into a conical flask or glass bottle.

[0344] 2. Gently plug the neck of the flask with Kimwipes. If using a glass bottle, loosen the stopper. Heat the suspension in a boiling water bath or microwave until the agarose is dissolved.

[0345] 3. Allow the clear solution to cool to 40-50°C, add 4S red staining solution and quickly cast the gel. When the gel is completely solidified, place it in the electrophoresis tank and add the newly prepared 1X TAE electrophoresis buffer until the gel is just covered.

[0346] 4. Prepare 10μL of virus, add 1.1μL 10X PCR Buffer, put into PCR instrument, 95℃ for 5 minutes, 72℃ for 5 minutes, 55℃ for 5 minutes, 37℃ for 5 minutes, 80℃ for 5 minutes, 72℃ for 5 minutes, 55℃ for 5 minutes, and 37℃ for 15 minutes.

[0347] 5. Add 0.2 volume of 6X gel loading buffer to the renatured sample.

[0348] 6. Add all samples dissolved in 6X loading buffer to the sample wells and start electrophoresis at 100V for 50 minutes.

[0349] 7. Imaging: Place the gel in a gel imager and take photos.

[0350] Figure 4 The results of neutral agarose gel electrophoresis of spAAV vectors packaged with cis packaging elements are shown. Lane MW: DNA molecular weight standard; Lane 1: spAAV vector packaged with ITR cis element; Lane 2: spAAV vector packaged with DtrsA′B′ cis element; Lane 3: spAAV vector packaged with trsA′B′ cis element; Lane 4: spAAV vector packaged with trsA′ cis element; Lane 5: spAAV vector packaged with DttRR cis element; Lane 6: spAAV vector packaged with ttRR cis element.

[0351] As can be seen from the figure, the spAAV vector DNA produced by packaging various cis packaging elements is a monopolar single-stranded DNA. When analyzed by neutral agarose gel electrophoresis, its DNA will appear as a DNA band that is much smaller than its molecular weight and often appears diffuse.

[0352] Then, DNA alkaline agarose gel electrophoresis analysis is performed, and the specific steps include:

[0353] 1. Preparation of alkaline agarose gel: Prepare agarose solution by adding 0.36g agarose powder and 27mL distilled water to a conical flask or glass bottle. Gently plug the neck of the conical flask with Kimwipes paper. If using a glass bottle, the stopper must be loosened. Heat the suspension in a microwave oven on medium heat until the agarose is dissolved. Allow the clear solution to equilibrate in a water bath at 56℃ for 5 minutes. Add 3mL 10X alkaline gel electrophoresis buffer (equilibrate in a water bath at 56℃ for 2 minutes), mix well, and quickly cast the gel. When the gel is completely solidified, place it in an electrophoresis tank and add freshly prepared 1X electrophoresis buffer (4℃) until the gel is just covered.

[0354] 2. Sample preparation: Take 30 μl of virus sample, add 3 μL of proteinase K (20 mg / mL), digest at 65°C for 15 minutes, centrifuge at 12000 g for 5 minutes, take 30 μL of supernatant, and add 6 μL of 6X alkaline gel loading buffer.

[0355] 3. Electrophoresis: Add all DNA samples to the sample wells and start electrophoresis at a voltage of <3.5V / cm (horizontal electrophoresis tank JY-SPAT 27V electrophoresis for 3h).

[0356] 4. Elution: Place the gel in 400 mL of water for injection and elute on a horizontal shaker at 56 rpm for 1 hour, changing the water every 30 minutes.

[0357] 5. Staining: Use 1X TAE staining solution containing 0.5 μg / mL EB (ethidium bromide) to stain the washed gel.

[0358] 6. Imaging: Place the gel in a gel imager and take photos.

[0359] Figure 5 The results of alkaline agarose gel electrophoresis of spAAV vectors are shown. Lane MW: DNA molecular weight standard; Lane 1: spAAV vector packaged with ITR cis element; Lane 2: spAAV vector packaged with DtrsA′B′ cis element; Lane 3: spAAV vector packaged with trsA′B′ cis element; Lane 4: spAAV vector packaged with trsA′ cis element; Lane 5: spAAV vector packaged with DttRR cis element; Lane 6: spAAV vector packaged with ttRR cis element.

[0360] As can be seen from the figure, the spAAV vector DNA produced by packaging various cis packaging elements is single-stranded, monopolar DNA. When analyzed by alkaline agarose gel electrophoresis, it presents a single DNA band, and its single-stranded DNA molecular weight is 4.7kb.

[0361] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A plurality of cis packaging elements for packaging single-stranded monopolar DNA recombinant adeno-associated viruses, wherein the cis packaging elements comprise a Rep binding site (RBS) and a Rep cleavage site (trs); The cis packaging element is capable of packaging its downstream spAAV genome into the AAV capsid protein, including when the cis packaging element is placed at the 5′ end of the packaged single-stranded monopolar DNA, the Rep protein packages the single-stranded monopolar DNA into the AAV capsid protein through recognition and cleavage.

2. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 1, characterized in that: The cis packaging element includes any one of the DNA elements of ITR, DtrsA′B′, trsA′B′, trsA′, DttRR, and ttRR; The 5′ end of the spAAV genome before packaging has the cis packaging element; The 3′ end of the spAAV genome before packaging does not have all or part of the DNA sequence of ITR and does not have other cis packaging elements.

3. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 2, characterized in that: The spAAV genome before packaging comprises the cis packaging element, exogenous nucleic acid and / or other exogenous DNA sequence in 5′ to 3′ order; The cis packaging element comprises RBE and trs; and / or, the cis packaging element comprises D, A′ and C′ segments derived from FlipITR; and / or, the cis packaging element comprises D, A′ and B′ segments derived from FlopITR; And / or, the Flip ITR has the nucleotide sequence shown in SEQ ID NO.1; And / or, the Flop ITR has the nucleotide sequence shown in SEQ ID NO.2; And / or, the RBE has the nucleotide sequence shown in SEQ ID NO.3; And / or, the trs has the nucleotide sequence shown in SEQ ID NO.4; and / or, the DtrsA′B′ has the nucleotide sequence shown in SEQ ID NO: 5; and / or, the trsA′B′ has the nucleotide sequence shown in SEQ ID NO: 6; and / or, the trsA′ has the nucleotide sequence shown in SEQ ID NO: 7; and / or, the DttRR has the nucleotide sequence shown in SEQ ID NO: 8; And / or, the ttRR has the nucleotide sequence shown in SEQ ID NO:

9.

4. The cis packaging element for packaging a single-stranded monopolar DNA recombinant adeno-associated virus according to claim 3, characterized in that: The exogenous nucleic acid is located downstream of the 5′ cis packaging element of the spAAV genome before packaging, and may have a nucleotide sequence for encoding an exogenous gene; Preferably, the exogenous nucleic acid comprises an exogenous gene expression cassette.

5. A spAAV vector, obtained by packaging a spAAV genome using the cis packaging element according to any one of claims 1 to 5; The spAAV includes an AAV capsid protein and a spAAV genome packaged by the AAV capsid protein; in, The spAAV genome packaged by the AAV capsid protein is derived from the spAAV genome before packaging; The spAAV genome comprises a spAAV vector of positive-strand DNA or a spAAV vector of negative-strand DNA, and the two are not produced at the same time.

6. The spAAV vector according to claim 5, characterized in that The 5′ end of the packaged spAAV genome in the spAAV vector has a cis packaging element; The exogenous nucleic acid is placed downstream of the 5′ end cis packaging element; And / or, the exogenous nucleic acid comprises a nucleotide sequence that may not encode an exogenous gene and / or has a nucleotide sequence for encoding an exogenous gene; And / or, the nucleotide sequence encoding the exogenous gene is an exogenous gene expression frame; And / or, the nucleotide sequence encoding the exogenous gene is a monopolar single-stranded DNA sequence; And / or, the nucleotide sequence not encoding the exogenous gene includes a stuffing sequence; the stuffing sequence may have different lengths, filling the final DNA length to reach 4.7 kb.

7. The spAAV vector according to claim 6, characterized in that The cis packaging element comprises an RBE sequence and a trs sequence, wherein the 5′ end sequence packaged into the spAAV virus capsid is the trs sequence portion remaining after Rep cleavage; And / or, the exogenous gene expression frame includes a promoter, an exogenous gene open reading frame and a poly A sequence in the 5′-3′ direction; Preferably, enhancers and introns may also be included between the promoter and the exogenous gene open reading frame; And / or, the exogenous gene expression frame includes a forward exogenous gene expression frame and a reverse exogenous gene expression frame; Preferably, the forward exogenous gene expression frame and the reverse exogenous gene expression frame are complementary sequences.

8. The spAAV vector according to claim 6, characterized in that The AAV capsid protein may be any one selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; and / or, the AAV capsid protein is an AAV capsid protein variant, including any one of a tyrosine single amino acid / multiple amino acid variant, a tyrosine variant, a serine variant, a threonine variant, an AAV-DJ multi-serotype chimera, or a polypeptide embedded variant; And / or, the AAV capsid protein can be produced by utilizing a plasmid expressing the AAV capsid protein Cap.

9. The spAAV vector according to any one of claims 5 to 8, characterized in that The spAAV vector can be produced by packaging the spAAV transgenic plasmid and the plasmid expressing the AAV capsid protein Cap and the replication protein Rep through an AAV vector packaging system, including an insect cell packaging system and a mammalian cell packaging system; The spAAV transgenic plasmid can be selected as the plasmid of the spAAV genome before packaging; Preferably, the spAAV transgenic plasmid is a pFastBacdual plasmid or a pFastBac1 plasmid; The spAAV transgenic plasmid can be designed as a positive-strand single-stranded DNA sequence or a negative-stranded DNA sequence encoding a foreign gene; the positive-stranded DNA sequence or the negative-stranded DNA sequence includes a cis packaging element, a promoter, a foreign gene open reading frame, and poly A in 5′ to 3′ order; And / or, the spAAV transgenic plasmid can produce the spAAV genome before packaging.

10. A method for obtaining the spAAV vector according to any one of claims 5 to 9, comprising transforming the spAAV transgenic plasmid, the Cap expression plasmid, and the Rep expression plasmid into competent E. coli cells respectively; selecting white colonies after at least one round of blue-white screening, amplifying and extracting the recombinant bacmid; transfecting insect cells with the recombinant bacmid to produce a recombinant baculovirus; and extracting the spAAV transgenic, Cap expression, and Rep expression recombinant baculoviruses, and co-infecting Sf9 cells with the recombinant baculoviruses to obtain the spAAV vector; in, The spAAV vector packaging system is an insect cell baculovirus packaging system or a mammalian cell packaging system.

11. A method for delivering exogenous nucleic acid to a cell, comprising contacting a cell with one or more spAAV vectors as described in any one of claims 5 to 9 and infecting the cell, wherein: The genome of the spAAV vector comprises the gene expression frame of the exogenous nucleic acid and / or other optional DNA sequences; the spAAV vector is integrated into the cell genome, homologous or non-homologous recombination occurs, and the exogenous gene expression is generated; The genome of the spAAV vector comprises the positive-strand single-stranded DNA sequence and / or the negative-strand single-stranded DNA sequence of the exogenous gene; The exogenous nucleic acid comprises a nucleic acid sequence of an exogenous gene or a nucleic acid sequence of a non-exogenous gene; Preferably, the spAAV vector comprises two or more spAAV vectors, which at least comprise a first spAAV vector having a positive-strand single-stranded DNA sequence of the exogenous gene and a second spAAV vector having a negative-strand single-stranded DNA sequence of the exogenous gene; Preferably, the first spAAV vector and the second spAAV vector are used alone or in combination; Preferably, the combined use comprises contacting the cell with the first spAAV vector and the second spAAV vector in sequence, so that the first spAAV vector and the second spAAV vector containing the positive-strand and negative-strand spAAV genomes, respectively, are rapidly complementary and paired in the nucleus of the host cell to form a double-stranded molecule, thereby initiating gene expression; And / or, the cell is a eukaryotic cell; preferably, the cell is an animal cell; more preferably, the cell includes a vertebrate cell or a mammalian cell.

12. Use of the cis packaging element according to any one of claims 1 to 4 in gene expression and / or gene therapy and / or gene editing and / or gene regulation and their products.

Images